The Human Immunodeficiency Virus (HIV) continues to be a major problem worldwide, even though a plethora of compounds have been approved for treatment. Due to the error-prone nature of viral reverse transcriptase and the high viral turnover (t1/2=1-3 days), the HIV genome mutates very rapidly. For example, reverse transcriptase is estimated to generate, on average, one mutation per replication of the 9.7 Kb genome that does not dramatically affect the ability of the virus to propagate. This leads to the formation of quasi-species, where many different mutants exist in a dynamic relationship.
To date, twenty-nine individual antiretroviral drugs from six drug classes have been approved by the U.S. Food and Drug Administration (FDA) to be used in the treatment of HIV-1 infection, including protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), non-nucleoside reverse transcriptase (NNRTI), integrase (INI), fusion (FI), and entry (EI), inhibitors. HIV-1 resistance to PI, NRTI, NNRTI, and INI can be determined using (i) indirect methods based on detection of specific amino acid substitutions (due to underlying nucleotide mutations) in the respective coding regions previously associated with resistance to specific antiretroviral drugs (i.e., genotyping), (ii) more direct methods that test the ability of a patient-derived virus to replicate in the presence of antiretroviral drugs in a cell-based assay (i.e., phenotyping), or (iii) a combination of both approaches that takes advantage of a large database to infer the level of HIV-1 drug resistance based on genotyping and its relationship with matched phenotypic data. Similarly, since treatment with CCR5 antagonists requires the prior knowledge of the HIV-1 coreceptor tropism in the patient, i.e., CCR5- or CXCR4-tropic viruses (R5 and X4, respectively), dual tropic (R5/X4), or a mixture of both R5 and X4 viruses, a multitude of phenotypic and genotypic approaches to determine HIV-1 coreceptor tropism have been developed. Phenotypic assays to determine HIV-1 drug resistance or tropism usually involve the generation of patient-derived pol- or env-recombinant viruses, respectively, to quantify their ability to infect susceptible cell lines expressing the appropriate HIV-1 receptors and coreceptors or, in the case of HIV-1 tropism, may also be based on the quantification of cell-to-cell fusion events. Whereas, genotypic HIV-1 tropism tests take advantage of the properties of specific regions in the env gene as determinants of CCR5 or CXCR4 tropism, mainly the V3 region of the gp120, and their interpretation based on a series of bioinformatic methods to infer the ability of HIV-1 to use any or both coreceptors to enter host cells.
As expected, phenotypic (experimental) and genotypic (computational) approaches to determine HIV-1 drug resistance or HIV-1 coreceptor tropism have some disadvantages, including the longer turnaround time and higher cost of the phenotypic assays or the intrinsic predictive nature of the genotypic tests. Particular emphasis has been made on the limited sensitivity of genotypic HIV-1 tropism assays to detect minor non-R5 variants, and to a lesser extent on the ability of genotypic HIV-1 drug resistance tests to detect minority drug resistant variants. In the case of HIV-1 drug resistance, the vast amount of information accumulated during the last two decades by correlating mutations with phenotypic data has led to the almost exclusive use of genotypic antiretroviral testing based on population (Sanger) sequencing to manage patients infected with HIV-1. In contrast, although several studies have shown significant concordance and similar predictive values, genotypic HIV-1 tropism assays based on population sequencing seem to be less sensitive and specific than phenotypic assays. Thus, a cell-based assay (Trofile, Monogram Biosciences) is currently the standard method to determine HIV-1 coreceptor tropism in the U.S., while genotypic HIV-1 tropism tests are largely used in Europe.
To date, all current commercial genotypic HIV-1 drug resistance assays are based on population sequencing, which can only detect minority variants present above 20% of the viral population. However, and although still uncertain, drug resistant HIV-1 minority variants (i.e., as low as 1% of the viral population) have been suggested to be clinically relevant as they have a high chance of being selected for under antiretroviral drug pressure.